SPRUCE BUDWORM (Choristoneura Fumiferana)T PHEROMONE CHEMISTRY and BEHAVIORAL RESPONSES to PHEROMONE COMPONENTS and ANALOGS

Journal of Chemical Ecology, Vol. 12, No. 2, 1986

SPRUCE BUDWORM (Choristoneura fumiferana)t PHEROMONE CHEMISTRY AND BEHAVIORAL RESPONSES TO PHEROMONE COMPONENTS AND ANALOGS

PETER J. SILK and L.P.S. KUENEN

Phe romone Research Group New Brunswick Research and Productivity Council P.O. Box 6000, College Hill Roarl Fredericton, New Brunswick E3B 5Hl

(Received June 3, 1985; accepted August 1, 1985)

Abstract-This paper reviews the sex pheromone chemistry and pheromone- mediated behavior of the spruce budworm and related coniferophagous (Choristoneura) budworms. ln C. funiferana, temporal changes in pheromone-gland monounsaturated fatty acids (pheromone precursors) enable the prediction of the primary sex pheromone components. This technique may also be applicable for predicting additional pheromone components. Tetra- decanal (14:Ald), previously shown to enhance close-range precopulatory behavior, lowers the threshold of response by males for upwind flight to a pheromone-component source. Spruce budworm males maintain upwind flight to 95 : 5 (El Z)-1, 12-pentadecadiene (diolefi n analog) after initiating upwind flight to a primary-component pheromone source (95 : 5 ElZll-|4: Ald). 'lhis is the first demonstration of apparently normal male flight responses to a pheromone analog.

Key Words-Cåoristoneura fumiferana, spruce budworm, Lepidoptera, Tortricidae, sex pheromone, behavior, flight tunnel, pheromone analog, pheromone fatty acid precursors.

INTRODUCTION

Lepidopteran female sex pheromones are, with few exceptions, multicompo- nent, consisting of a blend of two or more chemicals emitted at fairly consistent

I Lepidoptera: Tortricidae 367

0098-033 I /86/0200 0367$05.00/0 O I 986 Plcnum Publishing Corporat¡on 368 Srlrc eNo KusNeÀr

ratios and release rates. These chemical blends elicit in conspecific males se- quences of behaviors that include upwind flight which brings males to within close proximity of females. These behaviors are often readily observed in laboratory wind tunnels, and responses by males to synthetic pheromone sources can be compared to their responses to natural pheromone or females. The existence of a pheromone communication system in the spruce budworm (Choristoneura fumifurana Clemens) was demonstrated by Greenbank (1963), but elucidation of the blend of chemicals comprising the female sex pheromone is still incomplete. However, recent progress has been made in de- fining the sex-pheromone chemistry and pheromone-mediated behavior of this insect. This paper reviews the history and some current research related to the spruce budworm pheromone communication system.

BACKGROUND

In the genus Choristoneura (Lepidoptera: Tortricidae), known primary sex pheromone components are comprised of all-unsaturated c1a carbon-chain compounds. Different species' primary sex pheromone components have different oxygenated functional groups and varying blends of these components (see Inscoe, 1982). Specifically, the coniferophagous spruce budworms com- prise a group of closely related (choristoneurø) species, native to North Amer- ica (Freeman,196l; Freeman and Stehr, 1967; powell, 1980). Six species have been studied in terms of their pheromone specificity (sanders, l97l; Sanders et al., 1914, 1977). The components of these female-produced sex pheromones form a group of congeneric aldehydes (Al1-14: Ald), acetates (Â11- l4:Ac), and alcohols (^11-14:OH),^11-Cr4 with blends, geometrical isomer ratios, and release rates specific to each species (Table l). In attempting to characterize the sex pheromone of budworm species through cross-attraction studies, Sanders et al. (1977) concluded that c. fumiferana, c. occidentalis, and c. bíennis apparently had similar pheromones while C. pinus pinus, C. orae, and C. retiniana (: C. viridis; powell, 1980) were mutually cross-stimulating but did not appear to have sex pheromone components in common with the former group. This was an accurate assessment; subsequent research has shown that the former group utilizes al l-14 :Ald's as primary sex pheromone components, whereas the latter, utilize Âll-14:Ac's or Â11-14 : Aclall-14:oH blends (Table l). It has yet to be verified that the c. biennis sex pheromone is a1l*14: Ald, although present evidence is strongly supportive (Sanders, 1971; Sanders et al., 1914, lgil). Sex pheromone components of the remaining five species have been documented: C. fumifurøna (Sanders and Weatherston, 1916; Silk et al., 1980) and C. occidentalis (Cory etal.,7982; Silk et al., 1982) have been shown to release Elzlt-\4:Ald as SPRTJCE BUDWORM PHEROMONE 369

TaeLp l. Spx Pnpnovo¡re CovtpoNeNtso op Cot¡I¡sRoPHACous Choristoneura spp.

Species Primary Additional (Secondary) References

C. fumiferana El l-14:Ald Weatherston et al., 197 | 96:4 E/Zll-| :Ald Sanders and Weatherston, 1976 95:5 E/Zll-14:Ald, l4:Ald Silk et al., 1980 Alford et at., 1983 C. occidentalis 92:8 E/Z|I-|4:Ald Cory et al., 1982 92:8 E/Zll-14:Ald 89:ll E/Z Silk et al., 1982 I I - 14:Ac Alford and Silk, 1983 85:15 E/Z 1l-14:OH C. biennis E/ZlI-14:Aldb Sanders, 1971 E/Z ratio unknown Sanders et al., 19'74 C. orae 82:9:9, Gray et al., 1984 Ell-14:Ac, Zll-14:Ac, El l-14:OH C. pinus pinus 90:10, Silk et al., 1985b 85:15 E/Zll-14:Ac, 85:15 E/Zll-14:OH C. retiniana 92:8, E or Zll-|4:OH Daterman et al., 1984 E/Zll-14:Ac (enhances trap capture)

"E/Zll-\4:Ald : (E/Z)-ll-tetradecenal; E/Zll-l4:Ac : (E/Z)-11-tetradecenyl acetare. E/Z|1-14:OH : (E /Z) - 1 I -letradecen- I -ol ; I 4 : Ald : tetradecanal. áInferred from cross-attraction studies (Sanders, 197 l).

primary sex pheromone components. In contrast, C. pinus pinus (Silk et al., 1985b), C. retiniana (Daterman et al., 1984), and C. orae (Gray et al., 1984) release EIZ ll-14: Ac or Elzll-|4: AclElZll-14:OH blends as primary components (Table l). However, all species have Â11-14 : Ac in common as the major component in the pheromone gland (in references above). In C. fumifurana All-tetradecenyl acetate is synthesized de novo only in the pheromone gland, the Al1- 14: Ac is the direct biosynthetic precursor to the aldehyde pheromone (Morse and Meighen , 1984). Morse and Meighen (1984) also found that the female diel aldehyde-emission period was synchronous with acetate production, and this supports their hypothesis that in this (coniferophagous) Choristoneura group a metabolic relationship exists between the aldehyde, acetate, and alcohol. It seems likely, therefore, that species speciflcity in pheromone production is controlled by species-specific metabolic processes, giving rise to different functional groups, ratios, and release rates from the common acetate precursor. 3',70 Slr-r eNn KUeNEN

REVIEW OF SEX PHEROMONE CHEMISTRY OF SPRUCE BUDWORM

Among coniferophagous budworms, the spruce budworm, C. fumifurana, has been the most intensively studied. Early work indicated that spruce budworm females release a pheromone that "attracts" males (Greenbank, 1963). Subsequently , Ell-14: Ald was identified (Wearherston et al. , 197 l) as a pheromone component. The importance of adding the z to the E isomer was determined upon reanalysis of female volatiles and a (96:4) E/Zll-14:Ald blend was shown to maximize trap captures (Sanders and weatherston, lg76). solvent extracts of excised pheromone glands were inactive in eliciting male response (Sanders, 1971). Reanalysis of gland extracts identified 811-14:OH (Weath- erston and Maclean, 1974) and Ell-14:Ac (Wiesner et al., 1979), both of which inhibited trap capture (Sanders and Lucuik, 1972; Sanders etal.,1972; Sanders 1976). More detailed chemical analyses (Silk et al., 1980) of pheromone gland extracts showed that A1l-14:Ac (20-40 ng/insecr), (l-3 ng/insect), and Al l-14: OH (1-3 ng/insecr) were all presenr^11-14:Ald in 95 :5 EIZ raÍios; the saturated analogs of each functionality were also present at ca. I % of the corresponding E isomer. In contrast, effiuvia from "calling" females were found to contain Elzll-|4: Ald (95 :5; 10-40 nglinsect/nighr) and rhe saturated analog, tetradecanal (14:Ald) at ca.2% of the Z'll-14:Ald (Silk et at., l9g0). In addition, traces of (E)ll-14: Ac were found; no alcohols were detected. In the same study, field testing showed that there were no significant dif- ferences in trap captures between traps baited with all four components (formulated in PVC at female effiuvial ratios) compared to captures using the primary components (95 :5 ElZll-l4:Ald) alone. The reduction of trap capture by admixture of All-14:Ac with 95:5 ElZll-|4:Ald (Sanders et al., 1972) was confirmed; however, this effect appeared to be negated by the presence of 14: Ald, but only when all components were present in synthetic sources in female effiuvial ratios (silk et at., 1980). Pheromone release rate by "calling" females, measured by a specific and sensitive bioluminescent assay technique (Morse eta1.,7982, Meighen et al., 1981, 1982), occurs mainly during scotophase in a series of "bursts" at rates as high as 50 ng/hr with considerable individual variability.

REVIEW OF PHEROMONE-MEDIATED BEHAVIOR IN SPRUCE BUDV/ORM

Behavioral pattems involved in mate location in feral insects generally involve upwind flight, apparent close-range orientation, and copulation (Roelofs and carde, 1977). As in most moths, spruce budworm males locate conspecific females by flying upwind along a pheromone plume. Often, the effects of putative pheromone components on these male behaviors has been inferred from SPRUCE BUDWORM PHEROMONE 371 a chemical's effect on trap capture (until recently, this was also true for spruce budworm); however, observations and quantitation of some of these behaviors can be conducted in a sustained-flight wind tunnel (e.g., Miller and Roelofs, 1978). We review here recent field and wind-tunnel work, and present some recent progress. Sanders (198la) showed that synthetic 95:.5 ElZll'|4:Alds were equiv- alent in "attraction" to virgin females in field trapping experiments when rrsing a0.03% PVC source, which releases these primary components at a rate close to that of a "calling" virgin female (Silk et al., 1980). Furthermore, in preliminary wind-tunnel work, some males demonstrated an apparent full range of precopulatory behaviors, e.g., upwind flight, courtship, and copulatory at- tempts in response to only these two primary components (Sanders, 1979). However, more detailed observations in the wind tunnel (Sanders et aI., l98l) showed that males exposed to pheromone produced by "calling" females ex- hibited a higher incidence of upwind flight and made more rapid upwind progress than males exposed to a similar concentration of synthetic pheromone (95 :5 ElZll-14: Ald). Sanders and Seabrook (1982) concluded that it was unlikely that other chemicals were involved in the "attraction" phase of the mating process. How- ever, recent observations in our laboratory's wind tunnel and in the field (Alford et al., 1983) indicated an effect oftetradecanal (14:Ald) on the behaviorof male spruce budworm. A greater number of males initiated upwind flight and continued on to contact a source with ca. 5% 14:. Ald added to the Âl l-14: Ald's than when only the Al l-14: Alds were present. The addition of EII-14: Ac to the A1 1- 14 : Alds decreased the males' responsiveness to the aldehydes, but when present at low levels, its effect appeared to be attenuated when 14:Ald was also present (Alford et al., 1983). This latter effect was also seen in earlier field{rapping experiments (Silk et al., 1980) and was subsequently confirmed by further laboratory wind-tunnel studies (Sanders, 1984). In addition, Sanders (1984) found that duration of sustained flights was significantly longer in response to "calling" females than to any synthetic sources (with or without l4:Ald), implying that the synthetic blends are incomplete with respect to the female-emitted blend.

RECENT PROGRESS

It is apparent that not all the chemicals involved in the sex pheromone communication system of spruce budworm are known. In monitoring and mating disruption programs, however, it may not be essential to know every minor component although, as Roelofs has pointed out (1978), trap specificity and potency may be greatly increased as the synthetic lure more closely duplicates the natural pheromone, and it is presumed that the efficacy of mating disruption 3',12 SII-x eNo KueNeN would likewise be enhanced by a "more complete pheromone. " Sanders (1984) concluded that, although single components can cause considerable mating disruption in noctuids (campion et al., l98l), incomplete blends are considerably less effective against tortricids (Charlton and Cardé, l98l; Roelofs and Novak, 1981; Sanders, 198lb). This has led many to the conclusion that elucidation of the "complete" pheromone blend for budworm is of importance prior to further major mating-disruption tests. Recent work in our laboratory has, therefore, focused on the identification of the "complete" spruce budworm pheromone. Previous analyses of female spruce budworm effiuvia (Silk et al., 1980), using a Porapak@ Q collection technique, did not indicate, at least from a chemical perspective, the presence of other components. This method, however, does introduce relatively large amounts of contamination and minor components may have been obscured by this background contamination. The fact that, chemically, minor components are present in very low quan- tities in spruce budworm prompted the use of indirect techniques to ascertain component identity. Recently, Bjostad and Roelofs (1981, 1983) demonstrated that fatty acid precursors of female sex pheromone components can be identified and used to predict the presence of minor pheromone components (Bjostad and Roelofs, 1983; Bjostad et al., 1984). A technique for rapidly identifying these monounsaturated fatty acids in pheromone glands excised from female moths, using GC-MS analysis after dimethyldisulfide derivatizarion (DMDS) of gland extracts has been developed. A detailed analysis ofmonounsaturated fatty acids present in the pheromone glands of C. fumiferana, C. occidentalis, and C. pinus

TasLe 2. MoNouNsaruneren F¡rry Aclo Esrens OsrelNeo sv DMDS DBnrveuzettol op PnsrovoNe GI-¡N¡ Exrnacrs pnov THReg Choristoneura Specleso

DMDSå A- C. C. C. pinus Fatty ester fumiferana occidentalis pinus

9-12:Me T T T 5-14:Me VS VS T 7-14:Me T T T 9-14:Me VS VS VS 1l-14:Me M L S 7-16:Me M M S 9-16:Me L L L l1-16:Me M M S 12-16:Me T VS ND'

"Quantitative estimate relative to the most abundant fatty ester DMDS adduct (cr2-c16). L 100- 50; M 50-30; S 30-10; VS l0-1; T < I (L, M, S, VS, T: large, medium, smãlt, very smalt, and , trace, respectively; in Vo). Data from Dunkelblum et al., 19g5. 'Methanolyzed samples of chloroform-methanol extracts. 'ND, not detected. SPRUCE BUDWORM PHEROMONE 373 pinus was carried out; the techniques and results are discussed in detail else- where (Dunkelblum et al., 1985) and are summarized in Table 2. As can be seen, the major pheromone-gland components (which have been previously identified) are readily correlated with their corresponding fatty acids. For example, in these three budworm species, the ElZll-14:Me (ElZll-tetradece- noic acids, methyl esters) are correlated with the Al l-Cl4 moieties which com- prise the primary pheromone components (Tables I and 2). The fatty acid profile is very similar for all three species. The other unsaturated fatty acids may be precursors to as yet unidentified secondary sex pheromone components in these species. In some female moths, pheromone titer appears to be quite low on eclosion and increases to a maximum in a few days [e.g., Trichoplusiani Hübner (Shorey and Gaston, 1965)1. Based on this fact, analysis of monounsaturated fatty acids in spruce budworm females by the DMDS method was repeated with the view of ascertaining whether changes in fatty acid profile occurred with moth age. Pheromone glands were obtained from groups of ca. 2O females that were in the pupal stage (ust prior to emergence), t hr post-emergence, and 48 hr postemergence (all pupae and adults were maintained on a 16: 8 light-dark cycle and glands were excised during the 2nd and 3rd hr of scotophase, coincident with mature females' peak "calling" period). Pheromone glands (three repli- cates) excised from these three groups were extracted with chloroform-metha-

T¡.sLe 3. MoNouNserunerso Ferrv AcIo Esrens OsrrINe¡ sv DMDS DnnlvrrrzatloN or Extnecrs FRoM Pupel- ¡No 48-Houn PosrecLosIoN Spnuce Buowonv PsenovoNe GLaNoso

DMDSá A- Fatty ester Pupal glands' 48 h posteclosion

5-14:Me T T '1-1A:.Me T T 9-14:Me S, but largest s 14:Me 11-14:Me T M, both E/Zisomers present; largest 14:Me 7-16:Me S M 9-16:Me L,both E/Z; L, both E/Z isomers; largest l6:Me largest 16:Me I l-16:Me VS M l2-16:Me T ND'/

"Kuenen, Dunkelblum, and Silk, unpublished data. 'L, M, S, VS, and T same meaning as footnote in Table 3. 'Results for 1-h postemergence extracts were not significantly different from preemergence extracts and are not included in the table. ¿ND, not detected. 374 Sr-r eNo KueNreNr

nol (2: l) and subjected to acid-methanolysis followed by the DMDS reaction and capillary GC-MS (as in Dunkelblum et al., 1985). The results are presented in Table 3. A large increase is seen for Åll- 14:Me (from < l% to 3o-50%), and this is ro be expected since rhis fatty acyl moiety is most likely the direct biosynthetic precursor to al l-14: Ac and this component to the pheromone, Al1-14: Ald (Morse and Meighen, 1984). per- haps more significantly, a large increase in titer occurred with two other monounsaturated fatty acids: L7-16: Me and al l-16: Me with the latter showing the largest increase. This prompted the supposition, following the corollary of a1l- 14:Me + Â11-14:Ac - Ald, that a similar sequence of events may be occurring in the pheromone^11-14: gland of the spruce budworm for these Â16 components. Although this does not preclude the other fatty acids (and certainly only monounsaturated moieties would be detected by this method), these two components were considered a suitable starting point for further chemical and behavioral analyses. Reanalysis of female gland extracts (Dunkelblum and silk, unpublished data) indicated that, indeed, Al l-16: Ac is present in the spruce budworm sex pheromone gland (<0.1% of Ell-14:Ac) but that Â7-16:Ac could not be detected. However, neither al l-16: Ald nor l-16: Ac was detected in effiuvial material obtained by the Poropak Q collection^l method (Silk et al., 19g0), although background contamination probably precluded detection. Because Al l-14: Ac is the biosynthetic precursor to Al l-14:Ald (primary pheromone components), it was assumed, as a first step, that since Al1-16: Ac was determined as a gland component, it might be emitted as the aldehyde. However, initial wind-tunnel tests to determine the possible function of all-16:Ald's were inconclusive; further tests will be warranted if are found in female pheromone-gland volatiles. ^ll-16:Ald's The effects on behavior of putative pheromone components are usually assessed by adding them to compounds with known activity, (e.g., Linn and Gaston, 1981; Baker and cardé, 1979). Alternatively, changes in male behavior have been measured in response to removing one or more components from the believed complete volatile blend, or by giving males a choice between two merging odor plumes (e.g., Teal et al., 1986). we have recently tested another approach relating to male response to low dosages of pheromone blends. More specifically, we hypothesized that males would have a lower response threshold to "more complete" pheromone blends, i.e., the active space (Bossert and wilson' 1963) would become greater as the pheromone blend being tested more closely approached the natural female-emitted blend. To test this hypothesis in our sustained-flight wind tunnel, we employed two "pheromone" sources (Figure l). Both sources (rubber septa) were sus- pended, one behind the other, by thread (as in Baker and Kuenen, 1982; Kuenen and Baker, 1983) 15 cm above the center of the tunnel floor. The downwind SPRUCE BUDWORM PHEROMONE 375

I 2 s CLOTH 3 4 SCRTEN

lscreen "release " E AIR \ee E FLOT{ E

r90 I ¡lO 30 to o -2fJ distonce (cm) fron upvind sourcG (S)

Ftc. l. Schematic side view of sustained-flight wind tunnel. In "two-source" experiments maler were released from cages (at 1); as they flew upwind along the pheromone plume past (2) the down, wind source (4) was pulled quickly upward, and the males' subsequent flight behaviors to the upwinc source (5) were recorded.

source (DS) was constant at 3 or l0 ¡ig 95 :5 ElZll-l4:Ald, while the upwind source (US; the test source) ranged from 3 pCe5:5 ElZll-l4:Ald) down to 30 ng in halfJoglro¡ steps or the US was a blank. In a second experiment, the US contained 100 ng, 30 ng, or l0 ng Lll-14: Ald or the same three with 5% 14: Ald added. During the experiments, individual males were released from screen cages at the downwind end of the tunnel where some initiated upwind flight along the pheromone plume. When males. flying in or near the plume, *"r" i40 cm from the US, the DS was quickly raised to the top of the tunnel by pulling on its supporting thread. After raising of the DS, males were observed for their up- tunnel progress and scored for whether or not they flew to within 30 cm of the us or if they continued upwind flight and contacted the us. A test flight was considered to be over as soon as males flew out of the back of the tunnel or if they touched any of the tunnel's surfaces. Initially, we wanted to determine the threshold value for the us to which males continued to fly upwind after removal of the DS (10 ¡ig Âll-14:Ald). This threshold value was approximately 100 ng source dosage (Table 4). This is clearly seen by the reduction in the percentage of moths flying to within 30 cm of the US when the dosage was < 100 ng (Table 4; p < 0.05). Addition- ally, the time required for males to fly to within 30 cm of the us tended to increase when the US dosage was 30 ng or 100 ng Al l-14: Ald [Table 4; flight times were not analyzed statistically since numbers of moths flying close to the US was very low (l or 2) when the US contained a low pheromone dosel. 376 Sllr eNo KueueN

V/ith the threshold value determined using Lll-14: Ald alone, we next tested whether this threshold would be lowered by the addition of 5 % 14:. Ald to the US. Using identical procedures, we saw an apparent increased male responsiveness to low-dosage sources when tetradecanal was present. When dosage of the US was 100 ng All-14:Ald alone or plus 5% 14:Ald, approximately the same number of moths flew to within 30 cm of the US. Most of these moths reached this source and flight times were similar. At 30-ng source loadings, however, more males continued flight to the US when 5% 14: Ald was present (P < 0.05), and their flight times to both 30 cm-from-the-US and to the source were much less than flight times for males flying toward a source containing only the Al l-14: Ald's. (Table 5). It appears from this data that secondary sex pheromone components, which act to increase the number of males approaching a pheromone sourÇe, also lower the response threshold for flight continuation to a similar pheromone component blend after initiation of upwind flight.

PHEROMONE ANALOGS

In some moth species, structural analogs of pheromone components have shown biological activity in terms of mating disruption (e.g., Shorey et al. 1974; Carlson and Mclaughlin, 1982a,b). Activity of analogs has been predicated on the hypothesis that they are similar (isoelectronic or isosteric) to the natural compound(s) although these effects are difficult to predict. For the spruce bud- worn, for example, A9-dodecenyl formate, structurally very similar to 14: Ald, has proven inactive in the field (Sanders et al., 1980), although formate^11- analogs of the pheromones of the corn earworrn Heliothis zea (Boddie) (Mitch- ell et al., 1975) and the tobacco budworm Heliothis virescens (F.) (Beevor et al., 1977) are active mating disruptants. The response of insects to these pheromone analogs is generally difficult to assess except when examining for behaviors similar to those elicited by pheromones. Therefore, a simple and indirect assessment employing sticky traps has often been used to determine if the analogs (l) produce trap capture by themselves or (2) reduce or enhance trap capture when combined with known sex attractants, sex pheromones, or virgin females. Analogs have also been tested for their effect on mating (disruption) in field plots where analog sources have been distributed (e.g., Carlson and Mclaughlin, l982a,b). We recently reported biological activity of a diolefin analog 195:5 (E/Z)- 1,l2-pentadecadienel of All-14:Ald's (Silket al., 1985a). Briefly, the results were as follows. Diolefin formulated in PVC vial-caps produced trap capture by itself and enhanced trap capture when combined with the All-14:Ald's. rùy'hen formulated in PVC rods in a later test the same season, the diolefin produced little or no trap capture. However, when six PVCs (3.0% diolefin) were Ø

T¡.BI-B 4. RespoNsBs oF Mele Spnuce Buowonv ro DrrpeneNr Dosecns o¡ A-l l-I4:Aldo A¡ren Fucur INrrlauoN ro pg l0 Al l-14:Ald Plus lNorcerso Dos¡css or Al l-14:Ald oI

Dosage Al l-14:Ald on (US) the upwind source FI

Blank 30 ng 100 ng 300 ng lpg 3pg zo Mean % moths activatedá 86.6 (29.96) 100 (-) 96.0 (8.94) 88.0 (10.95) 100 (-) 96.0 (8.94) Mean % moths initiating flight 82.6 (29.0s) 96.0 (8.94) 92.0 (r0.9s) 88.0 (10.9s) 100 (-) 92.O (r7.89) Mean % moths flying upwind 58.6 (2 1 .90) 44.0 (16.73) 60.0 (20.00) 60.0 (24.49) 48.0 (22.8) s6.0 (21.91) Mean % moths flying to within 30 cm 8.0b (17.89) 12.0b (10.9s) 40.0a (14.14) 56.0a (26.08) 44.0a (26.08) 52.0a (17.89) of US' Mean Vo moths contacting US 4.0b (8.94) 8.0b (10.95) 40.Oa (14.14) 56.0a (26.08) 40.0a (24.49) 52.Oa (l'7.89) Flight duration from 140 cm to 30 cm 54.3 (12.84) 43.4 (20.60) 16.0 (3.06) 9.0 (1.88) 8.0 (2.59) 9.s (3.7r) downwind of US (sec)/ Flight duration from 140 cm s8.0 (4.4'7) s7.6 (4.24) 22.2 (4.99) 16.6 (4.86) 14.4 (s.31) r7.4 (5.7s) downwind, to the US (sec)d Mean closest approach to US (cm) 83.sa (20.3s) 49.sb (18.11) 22.9c (16.01) 9.3c (20.88) 4.0c (7.Is) l 8c (4.02) : "Al l-14:Ald 95:5 E/Zll-14:Ald. N : 25 (five groups of five); values are means (t SD) calculated from means of each group of five moths tested. óFirst three behavioral categories are indicators ofmale moth responsiveness used to assess the appropriateness ofevaluating the subsequent behaviors; therefore no analyses were conducted on these data. 'Means in each horizontal row having no letters in common are significantly different; P < 0.05, Duncan's new multiple-range test. 'Means were not analyzed since only-one or two moths flew to wiihin 30 cm or closer to the "pheromone" source at the lower dosages.

a ì-.¡ (t -l oo Tesr-E 5. RespoNsBs or M¡le Spnuce Bupwonu ro Tnnps Dosecns oF Al l-l4:Aldo .qNo THnsB Dos¡cns or Al l-\4:Ald + 57o 14:Ald Arren FucHr INru¡,uoN ro 3 pg A1l-I4:Ald Plus INolcereo Dos¡ces

Dosage Al l-14:Ald on the upwind source (US)

10ng+5% 30 ng + 57o lO0 ng + 5% 10 ng 30 ng 100 ng 14:Ald 14:Ald l4:Ald

Mean % moths activated' 100 (-) 100 (-) 96.0 (8.94) 92.0 (10.95) 100 (-) 100 (-) (8.94) lÑ.fea¡ Vo moths initiating 100 (-) r00 (-) 92.0 (10.9s) 84.0 (26.08) 96.0 (8.94) 96.0 flight Mean % moths flying s6.0 (29.66) 52.0 (17.89) 52.0 (30.33) 44.0 (8.94) 64.0 (26.08) 68.0 (30.33) upwind Mean % moths flying to 8.0c (10.95) 20.Oabc (14.14) 44.0ab (2r.91) 20.Obc (20.00) 32.Oabc (10.95) 52.0a (36.33) within 30 cm of US' Mean % moths contacting 4.0b (8.94) 4.0b (8.94) 44.0a (21 .91) 4.0b (8.94) 28.0a (10.95) 40.0a (37.42) US Flight duration from 140 26.6 (26.30) 49.9 (26.89) 19.0 (13.44) 25.s (22.4s) l6.s (8.86) ls.7 (6.60) cm to 30 cm, downwind of US (sec)/ Flight duration from 140 s2.'7 (-) 48.9 (-) 29.3 (18.98) 31.8 (-) 26.8 (lt.O2) 30.8 (22.31) cm downwind, to the US (sec)/ Mean closest approach to 49.0a (31.98) 43.lab (18.70) 6.7b (9.31) 43.lab (25.69) 27.0ab (19.58) t9.zab (24.00) US (cm) U) xr

"All details as in Table 4. Ëz áSee Table 4, footnote å. X 'See Table 4, footnote c. FI dsee Table 4, footnote d. z l¡ z SPRUCE BUDWORM PHEROMONE 379

Tesr-e 6. RpspoNses on Male Spnucp Bu¡wonv ro (1) Â11-14:Ald (2) DrolenrN ANaloc, eNo (3) 1: I Mlxrune oF BorH"

(l) l0 ngAll-l4:Ald (2) l0 ng DO (3) (l) + (2)

Mean % moths activated' 84.0a (16.80) 8.0b (l1.00) 92:0a (17.80) Mean % moths initiating flight 92.Oa (1"7.80) 36.0b (7.2O) 92.0a (11.00) Mean % moths flying upwind 48.0a (11.00) 4.0b (9.00) 48.0a (l 1.00) Mean distance flown per moth 150.8 (69.90) l8s (-) 150.8 (51.16) flying upwind (cm)'

"All-14:Ald :95:5 E/Z1l-14:Ald; DO : diolefin analog. N: 25 (five groups of five); values are means (f SD) calculated from means of each group of five moths tested. 'àMeans in each horizontal row having no letters in common are signiflcantly different; P < 0.05, Duncan's new multiple-range test. 'Means were not analyzed since only one moth flew up-tunnel (center column) when diolefin alone was present. placed in a circle around traps baited with All-14:Ald's, trap capture was reduced to a level not significantly different (P > 0.05) from traps surrounded by equally spaced PVCs containing0.O3% Lll-14:Ald's. From this we concluded that the diolefin had biological activity, but the trap capture data were ambiguous. Iùy'e then tested the diolefin in our wind tunnel. Male spruce budworm responses to (l) All-14:Ald's, (2) diolefin, and (3) I + 2 were examined. In measures of activation, flight initiation, flight distance, and percentage source contact, males responded similarly (Table 6; P > 0.05) to l-14:Ald's alone and when in admixrure (1 : 1) with the diolefin, but by itself^l the diolefin did not elicit upwind flight in males. [Although one male reached the upwind portion of the tunnel (Table 6), its flight path appeared random and was rarely in or near the analog plume.) In another study, male responses to the diolefin alone were not distinguishable (P > 0.05) from responses to a blank (control) source (Kuenen and Silk, unpublished data). Thus the diolefin did not enhance or diminish the responsiveness of males flying upwind toward a "pheromone" source. However, we reasoned that trap capture in diolefin-baited traps (mid-season study) may have occuffed after males responded to female-emitted pheromone, while no pheromone sources (virgin females) were present in our late season study where diolefin did not elicit male activity (trap capture). As a first test of this hypothesis, we allowed individual males to initiate flight to two chemical sources (see Figure 1). The upwind source contained diolefin alone or was blank, while the downwind (15 cm) source contained Lll-14:Ald's. After males had flown to within 140 cm of the upwind source, the downwind source was quickly pulled upward by its supporting thread. Males' progress was then recorded. Most notably, males continued their normal upwind ap- 380 Sllr eNo KueNeN proach to the diolefin source [upwind source; mean distance flown 163.3 cm * 45.73 (SD); N :24150 testedl, while, as expected, they soon initiated cross- wind casting (Kennedy and Marsh, 1974; Marsh et al., 1978) when the upwind source was blank [mean distance flown 119.0 cm + 29.75 (SD); P < 0.05; I test; N: 30/50 testedl. The males'flights to the diolefin source were not visually distinguishable from their flight responses to a Âll-14:Ald source, and 16 of the 24 moths flying upwind continued their upwind flight to reach and land on the diolefin source.

CONCLUSIONS

Progress in understanding the sex pheromone chemistry of the spruce budworm in relation to male behavior has been made in recent years. However, the sex pheromone blend of the budworm is not completely defined. Very low emission rates of additional pheromone components have made it difficult to identify these components. Identification of pheromone gland fatty acids, and particu- larly their temporal variation in relation to pheromone production, may become a very useful technique in identifying these additional components. The data we have presented on male flight thresholds is not definitive at this point, but represents an approach to the study of the spruce budworm communication system. Our analyses of male responses to a pheromone analog are also preliminary; however, the demonstration of normal male upwind flight in response to the diolefin analog is, to the best of our knowledge, the first report of the observation of this behavioral response to a pheromone analog.

Acknowledgmen¡s-This research was funded in part by the New Brunswick Department of Natural Resources, and the Canadian Forestry Service. We thank Dr. C. Northcott (RPC) for synthesis of Al 1-16: Ald and the diolefin analog from a nonaldehyde source, and Dr. G. Lonergan (UNB) for synthesis of the diolefin from Al l-14: Ald. We also thank Dr. E. Butterworth and M. McClure for technical assistance and L. Jewett for typing the manuscript. Special thanks go to Dr. E. Dunkelblum, for his expertise and enthusiasm during a sabbatical year from the Volcani Center, Bet Dagan, Israel during 1984-85 with PRG/RPC.

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